H3 influenza a virus

ABSTRACT

The invention provides an isolated H3 equine influenza A virus, as well as methods of preparing and using the virus, and genes or proteins thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/255,719, filed Apr. 17, 2014, which is a continuation of U.S. patentapplication Ser. No. 13/839,111, filed Mar. 15, 2013, which is acontinuation of U.S. patent application Ser. No. 12/503,712, filed Jul.15, 2009, which is a divisional of U.S. patent application Ser. No.11/033,248, filed Jan. 11, 2005, which application is incorporatedherein by reference.

STATEMENT OF GOVERNMENT RIGHTS

The invention was made, at least in part, with a grant from theGovernment of the United States of America (grant 2001-35204-10184 fromthe United States Department of Agriculture). The Government may havecertain rights to the invention.

BACKGROUND

Influenza is a major respiratory disease in some mammals includinghorses and is responsible for substantial morbidity and economic losseseach year. In addition, influenza virus infections can cause severesystemic disease in some avian species, leading to death. The segmentednature of the influenza virus genome allows for reassortment of segmentsduring virus replication in cells infected with two or more influenzaviruses. The reassortment of segments, combined with genetic mutationand drift, can give rise to a myriad of divergent strains of influenzavirus over time. The new strains exhibit antigenic variation in theirhemagglutinin (HA) and/or neuraminidase (NA) proteins, and in particularthe gene coding for the HA protein has a high rate of variability. Thepredominant current practice for the prevention of flu is vaccination.Most commonly, whole virus vaccines are used. As the influenza HAprotein is the major target antigen for the protective immune responsesof a host to the virus and is highly variable, the isolation ofinfluenza virus and the identification and characterization of the HAantigen in viruses associated with recent outbreaks is important forvaccine production. Based on prevalence and prediction, a vaccine isdesigned to stimulate a protective immune response against thepredominant and expected influenza virus strains (Park et al., 2004).

There are three general types of influenza viruses, Type A, Type B andType C, which are defined by the absence of serological crossreactivitybetween their internal proteins. Influenza Type A viruses are furtherclassified into subtypes based on antigenic and genetic differences oftheir glycoproteins, the HA and NA proteins. All the known HA and NAsubtypes (H1 to H15 and N1 to N9) have been isolated from aquatic birds,which are though to act as a natural reservoir for influenza. H7N7 andH3N8 Type A viruses are the most common causes of equine influenza, andthose subtypes are generally incorporated into equine influenzavaccines.

Thus, there is a continuing need to isolate new influenza virusisolates, e.g., for vaccine production.

SUMMARY OF THE INVENTION

The invention provides isolated H3 equine derived influenza type A virusthat was isolated from a foal that succumbed to a fatal pneumonia, whichvirus has characteristic substitutions at residues 78 and 159 of HA(numbering of positions is that in the mature protein which lacks a 15amino acid signal peptide), i.e., the residue at position 78 of HA isnot valine and the residue at position 159 is not asparagine. In oneembodiment, the isolated H3 influenza A virus of the invention has aconservative substitution at residue 78, e.g., a valine to an alaninesubstitution, and a nonconservative substitution at residue 159, e.g.,an asparagine to a serine substitution. In one embodiment, the isolatedH3 influenza A virus of the invention has a residue other thanmethionine at position 29, e.g., a nonconservative substitution, aresidue other than lysine at position 54, e.g., a nonconservativesubstitution, a residue other than serine at position 83, e.g., anonconservative substitution, a residue other than asparagine atposition 92, e.g., a nonconservative substitution, a residue other thanleucine at position 222, e.g., a nonconservative substitution, a residueother than alanine at position 272, e.g., a conservative substitution,and/or a residue other than threonine at position 328, e.g., aconservative substitution. Conservative amino acid substitutions referto the interchangeability of residues having similar side chains. Forexample, a group of amino acids having aliphatic side chains is glycine,alanine, valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine and tryptophan; a group of amino acids having basic side chainsis lysine, arginine and histidine; and a group of amino acids havingsulfur-containing side chain is cysteine and methionine. In oneembodiment, conservative amino acid substitution groups are:threonine-valine-leucine-isoleucine-alanine; phenylalanine-tyro sine;lysine-arginine; alanine-valine; glutamic-aspartic; andasparagine-glutamine.

In one embodiment, the influenza virus of the invention includes one ormore viral proteins (polypeptides) having substantially the same aminoacid sequence as one of SEQ ID NOs:1-8, 17 and/or 18, so long as the HAhas the characteristic substitutions at residues 78 and 159. An aminoacid sequence which is substantially the same as a reference sequencehas at least 95%, e.g., 96%, 97%, 98% or 99%, amino acid sequenceidentity to that reference sequence, and may include sequences withdeletions, e.g., those that result in a deleted viral protein havingsubstantially the same activity or capable of being expressed atsubstantially the same level as the corresponding full-length, matureviral protein, insertions, e.g., those that result in a modified viralprotein having substantially the same activity or capable of beingexpressed at substantially the same level as the correspondingfull-length, mature viral protein, and/or substitutions, e.g., thosethat result in a viral protein having substantially the same activity orcapable of being expressed at substantially the same level as thereference protein. In one embodiment, the one or more residues which arenot identical to those in the reference sequence may be conservative ornonconservative substitutions which one or more substitutions do notsubstantially alter the expressed level or activity of the protein withthe substitution(s), and/or the level of virus obtained from a cellinfected with a virus having that protein. As used herein,“substantially the same expressed level or activity” includes adetectable protein level that is about 80%, 90% or more, the proteinlevel, or a measurable activity that is about 30%, 50%, 90%, e.g., up to100% or more, the activity, of a full-length mature polypeptidecorresponding to one of SEQ ID NOs:1-8, 17 or 18. In one embodiment, thevirus comprises a polypeptide with one or more, for instance, 2, 5, 10,15, 20 or more, amino acid substitutions, e.g., conservativesubstitutions of up to 5% of the residues of the full-length, matureform of a polypeptide having SEQ ID NOs:1-8, 17 or 18. The isolatedvirus of the invention may be employed alone or with one or more othervirus isolates, e.g., other influenza virus isolates, in a vaccine, toraise virus-specific antisera, in gene therapy, and/or in diagnostics.Accordingly, the invention provides host cells infected with the virusof the invention, and isolated antibody specific for the virus.

The invention also provides an isolated nucleic acid molecule(polynucleotide) comprising a nucleic acid segment corresponding to atleast one of the proteins of the virus of the invention, a portion ofthe nucleic acid segment for a viral protein having substantially thesame level or activity as a corresponding polypeptide encoded by one ofSEQ ID NOs:1-8, 17 or 18, or the complement of the nucleic acidmolecule. In one embodiment, the isolated nucleic acid molecule encodesa polypeptide which has substantially the same amino acid sequence,e.g., has at least 95%, e.g., 96%, 97%, 98% or 99%, contiguous aminoacid sequence identity to a polypeptide having one of SEQ ID NOs:1-8, 17or 18. In one embodiment, the isolated nucleic acid molecule comprises anucleotide sequence which is substantially the same as, e.g., has atleast 50%, e.g., 60%, 70%, 80% or 90% or more, contiguous nucleic acidsequence identity to, one of SEQ ID NOs:9-16, or the complement thereof,and encodes a polypeptide having at least 95%, e.g., 96%, 97%, 98% or99%, contiguous amino acid sequence identity to a polypeptide having oneof SEQ ID NOs:1-8, 17 or 18.

The isolated nucleic acid molecule of the invention may be employed in avector to express influenza proteins, e.g., for recombinant proteinvaccine production or to raise antisera, as a nucleic acid vaccine, foruse in diagnostics or, for vRNA production, to prepare chimeric genes,e.g., with other viral genes including other influenza virus genes,and/or to prepare recombinant virus, e.g., see Neumann et al. (1999)which is incorporated by reference herein. Thus, the invention alsoprovides isolated viral polypeptides, recombinant virus, and host cellscontacted with the nucleic acid molecule(s) and/or recombinant virus ofthe invention, as well as isolated virus-specific antibodies, forinstance, obtained from mammals infected with the virus or immunizedwith an isolated viral polypeptide or polynucleotide encoding one ormore viral polypeptides.

The invention further provides at least one of the following isolatedvectors, for instance, one or more isolated influenza virus vectors, ora composition comprising the one or more vectors: a vector comprising apromoter operably linked to an influenza virus PA DNA for a PA havingsubstantially the same amino acid sequence as SEQ ID NO:5 linked to atranscription termination sequence, a vector comprising a promoteroperably linked to an influenza virus PB1 DNA for a PB1 havingsubstantially the same amino acid sequence as SEQ ID NO:3 linked to atranscription termination sequence, a vector comprising a promoteroperably linked to an influenza virus PB2 DNA for a PB2 havingsubstantially the same amino acid sequence as SEQ ID NO:4 linked to atranscription termination sequence, a vector comprising a promoteroperably linked to an influenza virus HA DNA for a HA havingsubstantially the same amino acid sequence as SEQ ID NO:1 linked to atranscription termination sequence, a vector comprising a promoteroperably linked to an influenza virus NP DNA for a NP havingsubstantially the same amino acid sequence as SEQ ID NO:6 linked to atranscription termination sequence, a vector comprising a promoteroperably linked to an influenza virus NA DNA for a NA havingsubstantially the same amino acid sequence as SEQ ID NO:2 linked to atranscription termination sequence, a vector comprising a promoteroperably linked to an influenza virus M DNA for a M a havingsubstantially the same amino acid sequence as SEQ ID NO:7 (M1) and/orSEQ ID NO:17 (M2), linked to a transcription termination sequence,and/or a vector comprising a promoter operably linked to an influenzavirus NS DNA for a NS having substantially the same amino acid sequenceas SEQ ID NO:8 (NS1) and/or SEQ ID NO:18 (NS2), linked to atranscription termination sequence. Optionally, two vectors may beemployed in place of the vector comprising a promoter operably linked toan influenza virus M DNA linked to a transcription termination sequence,e.g., a vector comprising a promoter operably linked to an influenzavirus M1 DNA linked to a transcription termination sequence and a vectorcomprising a promoter operably linked to an influenza virus M2 DNAlinked to a transcription termination sequence. Optionally, two vectorsmay be employed in place of the vector comprising a promoter operablylinked to an influenza virus NS DNA linked to a transcriptiontermination sequence, e.g., a vector comprising a promoter operablylinked to an influenza virus NS1 DNA linked to a transcriptiontermination sequence and a vector comprising a promoter operably linkedto an influenza virus NS2 DNA linked to a transcription terminationsequence. An influenza virus vector is one which includes at least 5′and 3′ noncoding influenza virus sequences.

Hence, the invention provides vectors, e.g., plasmids, which encodeinfluenza virus proteins, and/or encode influenza vRNA, both native andrecombinant vRNA. Thus, a vector of the invention may encode aninfluenza virus protein (sense) or vRNA (antisense). Any suitablepromoter or transcription termination sequence may be employed toexpress a protein or peptide, e.g., a viral protein or peptide, aprotein or peptide of a nonviral pathogen, or a therapeutic protein orpeptide. In one embodiment, to express vRNA, the promoter is a RNApolymerase I promoter, a RNA polymerase II promoter, a RNA polymeraseIII promoter, a T3 promoter or a T7 promoter. Optionally the vectorcomprises a transcription termination sequence such as a RNA polymeraseI transcription termination sequence, a RNA polymerase II transcriptiontermination sequence, a RNA polymerase III transcription terminationsequence, or a ribozyme.

A composition of the invention may also comprise a gene or open readingframe of interest, e.g., a foreign gene encoding an immunogenic peptideor protein useful as a vaccine. Thus, another embodiment of theinvention comprises a composition of the invention as described above inwhich one of the influenza virus genes in the vectors is replaced with aforeign gene, or the composition further comprises, in addition to allthe influenza virus genes, a vector comprising a promoter linked to 5′influenza virus sequences linked to a desired nucleic acid sequence,e.g., a cDNA of interest, linked to 3′ influenza virus sequences linkedto a transcription termination sequence, which, when contacted with ahost cell permissive for influenza virus replication optionally resultsin recombinant virus. In one embodiment, the DNA of interest is in anantisense orientation. The DNA of interest, whether in a vector for vRNAor protein production, may encode an immunogenic epitope, such as anepitope useful in a cancer therapy or vaccine, or a peptide orpolypeptide useful in gene therapy.

A plurality of the vectors of the invention may be physically linked oreach vector may be present on an individual plasmid or other, e.g.,linear, nucleic acid delivery vehicle.

The invention also provides a method to prepare influenza virus. Themethod comprises contacting a cell, e.g., an avian or a mammalian cell,with the isolated virus of the invention or a plurality of the vectorsof the invention, e.g., sequentially or simultaneously, for example,employing a composition comprising a plurality of the vectors, in anamount effective to yield infectious influenza virus. The invention alsoincludes isolating virus from a cell infected with the virus orcontacted with the vectors and/or composition. The invention furtherprovides a host cell infected with the virus of the invention orcontacted with the composition or vectors of the invention. In oneembodiment, a host cell is infected with an attenuated (e.g., coldadapted) donor virus and a virus of the invention to prepare acold-adapted reassortant virus useful as a cold-adapted live virusvaccine.

The invention also provides a method to induce an immune response in amammal, e.g., to immunize a mammal, against one more pathogens, e.g.,against a virus of the invention and optionally a bacteria, a differentvirus, or a parasite or other antigen. An immunological response to acomposition or vaccine is the development in the host organism of acellular and/or antibody-mediated immune response to a viralpolypeptide, e.g., an administered viral preparation, polypeptide or oneencoded by an administered nucleic acid molecule, which can prevent orinhibit infection to that virus or a closely (structurally) relatedvirus. Usually, such a response consists of the subject producingantibodies, B cell, helper T cells, suppressor T cells, and/or cytotoxicT cells directed specifically to an antigen or antigens included in thecomposition or vaccine of interest. The method includes administering tothe host organism, e.g., a mammal, an effective amount of the influenzavirus of the invention, e.g., an attenuated, live virus, optionally incombination with an adjuvant and/or a carrier, e.g., in an amounteffective to prevent or ameliorate infection of an animal such as amammal by that virus or an antigenically closely related virus. In oneembodiment, the virus is administered intramuscularly while in anotherembodiment, the virus is administered intranasally. In some dosingprotocols, all doses may be administered intramuscularly orintranasally, while in others a combination of intramuscular andintranasal administration is employed. The vaccine may further containother isolates of influenza virus including recombinant influenza virus,other pathogen(s), additional biological agents or microbial components,e.g., to form a multivalent vaccine. In one embodiment, intranasalvaccination with inactivated equine influenza virus and a mucosaladjuvant, e.g., the non-toxic B chain of cholera toxin, may inducevirus-specific IgA and neutralizing antibody in the nasopharynx as wellas serum IgG.

The equine influenza vaccine may employed with other anti-virals, e.g.,amantadine, rimantadine, and/or neuraminidase inhibitors, e.g., may beadministered separately in conjunction with those anti-virals, forinstance, administered before, during and/or after.

Further provided is a diagnostic method which employs a virus of theinvention, an isolated viral protein encoded thereby, or antiseraspecific for the virus or protein, to detect viral specific antibodiesor viral specific proteins.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Sequences of A/Equine/Wisconsin/1/03. SEQ ID NOs:1-8, 17 and 18represent the deduced amino acid sequence for HA, NA, PB1, PB2, PA, NP,M1, NS1, M2, and NS2, respectively, of A/Equine/Wisconsin/1/03. SEQ IDNOs:9-16 represent the mRNA sense nucleotide sequence for HA, NA, PB1,PB2, PA, NP, M (M1 and M2) and NS (NS1 and NS2), respectively, ofA/Equine/Wisconsin/1/03.

FIG. 2. Sequence alignment of HA-1 of A/Equine/NewYork/99 (SEQ ID NO:19)and A/Equine/Wisconsin/1/03 (SEQ ID NO:20).

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “isolated” refers to in vitro preparationand/or isolation of a nucleic acid molecule, e.g., vector or plasmid,peptide or polypeptide (protein), or virus of the invention, so that itis not associated with in vivo substances, or is substantially purifiedfrom in vitro substances. An isolated virus preparation is generallyobtained by in vitro culture and propagation, and is substantially freefrom other infectious agents.

As used herein, “substantially purified” means the object species is thepredominant species, e.g., on a molar basis it is more abundant than anyother individual species in a composition, and preferably is at leastabout 80% of the species present, and optionally 90% or greater, e.g.,95%, 98%, 99% or more, of the species present in the composition.

As used herein, “substantially free” means below the level of detectionfor a particular infectious agent using standard detection methods forthat agent.

A “recombinant” virus is one which has been manipulated in vitro, e.g.,using recombinant DNA techniques, to introduce changes to the viralgenome.

As used herein, the term “recombinant nucleic acid” or “recombinant DNAsequence or segment” refers to a nucleic acid, e.g., to DNA, that hasbeen derived or isolated from a source, that may be subsequentlychemically altered in vitro, so that its sequence is not naturallyoccurring, or corresponds to naturally occurring sequences that are notpositioned as they would be positioned in the native genome. An exampleof DNA “derived” from a source, would be a DNA sequence that isidentified as a useful fragment, and which is then chemicallysynthesized in essentially pure form. An example of such DNA “isolated”from a source would be a useful DNA sequence that is excised or removedfrom said source by chemical means, e.g., by the use of restrictionendonucleases, so that it can be further manipulated, e.g., amplified,for use in the invention, by the methodology of genetic engineering.

Influenza Virus Type A Structure and Propagation

Influenza A viruses possess a genome of eight single-strandednegative-sense viral RNAs (vRNAs) that encode at least ten proteins. Theinfluenza virus life cycle begins with binding of the hemagglutinin (HA)to sialic acid-containing receptors on the surface of the host cell,followed by receptor-mediated endocytosis. The low pH in late endosomestriggers a conformational shift in the HA, thereby exposing theN-terminus of the HA2 subunit (the so-called fusion peptide). The fusionpeptide initiates the fusion of the viral and endosomal membrane, andthe matrix protein (M1) and RNP complexes are released into thecytoplasm. RNPs consist of the nucleoprotein (NP), which encapsidatesvRNA, and the viral polymerase complex, which is formed by the PA, PB1,and PB2 proteins. RNPs are transported into the nucleus, wheretranscription and replication take place. The RNA polymerase complexcatalyzes three different reactions: synthesis of an mRNA with a 5′ capand 3′ polyA structure, of a full-length complementary RNA (cRNA), andof genomic vRNA using the cRNA as a template. Newly synthesized vRNAs,NP, and polymerase proteins are then assembled into RNPs, exported fromthe nucleus, and transported to the plasma membrane, where budding ofprogeny virus particles occurs. The neuraminidase (NA) protein plays acrucial role late in infection by removing sialic acid fromsialyloligosaccharides, thus releasing newly assembled virions from thecell surface and preventing the self aggregation of virus particles.Although virus assembly involves protein-protein and protein-vRNAinteractions, the nature of these interactions is largely unknown.

Any cell, e.g., any avian or mammalian cell, such as a human, canine,bovine, equine, feline, swine, ovine, mink, e.g., MvLu1 cells, ornon-human primate cell, including mutant cells, which supports efficientreplication of influenza virus can be employed to isolate and/orpropagate influenza viruses. Isolated viruses can be used to prepare areassortant virus, e.g., an attenuated virus. In one embodiment, hostcells for vaccine production are those found in avian eggs. In anotherembodiment, host cells for vaccine production are continuous mammalianor avian cell lines or cell strains. It is preferred to establish acomplete characterization of the cells to be used, so that appropriatetests for purity of the final product can be included. Data that can beused for the characterization of a cell includes (a) information on itsorigin, derivation, and passage history; (b) information on its growthand morphological characteristics; (c) results of tests of adventitiousagents; (d) distinguishing features, such as biochemical, immunological,and cytogenetic patterns which allow the cells to be clearly recognizedamong other cell lines; and (e) results of tests for tumorigenicity.Preferably, the passage level, or population doubling, of the host cellused is as low as possible.

It is preferred that the virus produced by the host cell is highlypurified prior to vaccine or gene therapy formulation. Generally, thepurification procedures result in the extensive removal of cellular DNA,other cellular components, and adventitious agents. Procedures thatextensively degrade or denature DNA can also be used.

Equine Influenza Virus Detection

Disease causing equine influenza viruses are generally Type A influenzaviruses of the H7N7 (equi-1) and H3N8 (equi-2) subtypes. These generallydiffer from the subtypes that cause infection in man (H1N1, H2N2 andH3N2). Equine influenza is contracted by either inhalation or contactwith secretions (e.g., physiological fluid) containing live virus. Thevirus infects the epithelial cells of the upper and lower airways andcan cause deciliation of large areas of the respiratory tract within 4to 6 days. As a result, the mucociliary clearance mechanism iscompromised and tracheal clearance rates may be reduced for up to 32days following infection. Bronchitis and bronchiolitis develop followedby interstitial pneumonia accompanied by congestion, edema and leukocyteinfiltration. In general, H3N8 viruses cause more severe disease thanH7N7 viruses; viruses of the H3N8 subtype are more pneumotropic and havealso been associated with myocarditis.

Clinical signs in previously influenza-naïve animals are easilyrecognizable. Influenza is characterized by its sudden onset with anincubation period of 1 to 3 days. The first sign is an elevation of bodytemperature (up to 41° C.), which is usually biphasic. This is followedby a deep dry cough that releases large quantities of virus into theatmosphere often accompanied by a serous nasal discharge, which maybecome mucopurulent due to secondary bacterial infection. The other mostcommonly observed clinical signs are myalgia, inappetance, and enlargedsubmandibular lymph nodes. Edema of the legs and scrotum is observedvery rarely. The severity of the disease varies with the dose and strainof virus and the immune status of the horse.

Previously healthy, immunocompetent adult horses usually recover fromuncomplicated influenza within 10 days, although coughing may persistfor longer. If secondary bacterial infection occurs, it can prolong therecovery period. However, relatively high mortality rates have beenrecorded in foals, animals in poor condition and donkeys. If maternalantibody is absent at the time of exposure, young foals may develop aviral pneumonia leading to death. Deaths among adult animals are usuallya consequence of secondary bacterial infection leading to pleuritis,suppurative pneumonia or rarely, purpura haemorrhagica. Sequelae ofequine influenza can include chronic pharyngitis, chronic bronchiolitis,myocarditis, and alveolar emphysema, which can contribute to heaves, andsecondary sinus and guttural pouch infections.

Clinical signs in animals partially immune as a result of vaccination orprevious infection are more difficult to recognize as there may belittle or no coughing or pyrexia. Whereas spread of infection throughouta group of naïve animals is always rapid, there have been outbreaks inwhich the infection circulated subclinically in vaccinated horses for 18days before inducing recognizable clinical signs.

Outbreaks of infectious respiratory disease may be caused by variousagents, including equine herpes viruses, rhinoviruses, adenoviruses, andarteritis viruses, Streptococcus equi, or S. zooepidemicus. Apresumptive diagnosis of influenza based on clinical signs should beconfirmed by virus isolation or detection, or by serological testing.Laboratory confirmation of a clinical diagnosis may be by traditionalisolation of virus from nasopharyngeal swabs or serology to demonstrateseroconversion, or by rapid diagnostic tests which detect the presenceof viral antigens, viral nucleic acid, or virally infected cells inrespiratory secretions. Rapid diagnostic tests, despite theirconvenience and ease of use, provide little or no information aboutgenetic or antigenic characteristics of the infecting strain of virusand do not allow isolation of the virus.

Nasopharyngeal swabs for virus isolation or detection should be taken aspromptly as possible. Results of experimental challenge studies suggestthat peak viral titers are obtained during the initial 24 to 48 hours offever, on the second or third day after infection, and the duration ofviral shedding is usually not more than 4 or 5 days. Nasal swab samplesare taken by passing a swab as far as possible into the horse'snasopharynx via the ventral meatus to absorb respiratory secretions.Swabs should be transferred immediately to a container with virustransport medium and transported on ice to maintain viability of thevirus. Virus is unlikely to survive if dry swabs are taken and there isan increased chance of contamination if bacterial transport medium isused. Nasal swab samples may be inoculated into the allantoic (oramniotic) cavity of 9- to 11-day-old embryonated hens' eggs. Afterincubation at 33-35° C. for 3 days, the allantoic fluid is harvested andtested for haemagglutinating activity. Alternatively, cell culture maybe used to isolate viruses. Influenza infection can also be diagnosed bycomparison of the results of serological testing of an acute serumsample taken as soon as possible after the onset of clinical signs and aconvalescent serum sample taken 2 to 4 weeks later.

The haemagglutination inhibition (HI) test measures the capacity ofinfluenza-specific antibody present in serum samples to inhibit theagglutination of red blood cells by virus. Sera are heat-inactivated andpre-treated to reduce non-specific reactions and serially diluted priorto incubation with a standard dose of virus in a U-bottomed microtiterplate. A suspension of red blood cells is added and, after a furtherincubation period, examined for agglutination. A four-fold rise invirus-specific antibodies indicates infection. Whole virus antigen maybe used for H7N7 viruses, but Tween 80-ether disrupted antigen isusually required to enhance the sensitivity of the assay for H3N8viruses. In repeatedly vaccinated horses, infection may fail tostimulate a 4-fold increase in HI titer.

The single-radial haemolysis (SRH) test, although less strain-specific,is more reproducible and less error prone than the HI test and, as it isa linear test, is more sensitive, enabling detection of smallerincreases in antibody induced by infection in heavily vaccinated horses.The SRH test is based on the ability of influenza-specific antibodies tolyse virus-coated red blood cells in the presence of complement. Testsera are added to wells punched in agarose containing coated red bloodcells and complement and allowed to diffuse through the agarose for 20hours. The areas of clear zones of haemolysis around the wells areproportional to the level of influenza antibody present in the serumsamples.

If horses are vaccinated in the face of infection, it may not bepossible, using the HI and SRH assays, to determine whether any increasein antibody levels is due to vaccination or infection.

Influenza Vaccines

A vaccine of the invention includes an isolated influenza virus of theinvention, and optionally one or more other isolated viruses includingother isolated influenza viruses, West Nile virus, equine herpes virus,equine arteritis virus, equine infectious anemia lentivirus, rabiesvirus, Eastern and/or Western and/or Venezuelan equine encephalitisvirus, one or more immunogenic proteins or glycoproteins of one or moreisolated influenza viruses or one or more other pathogens, e.g., animmunogenic protein from one or more bacteria, non-influenza viruses,yeast or fungi, or isolated nucleic acid encoding one or more viralproteins (e.g., DNA vaccines) including one or more immunogenic proteinsof the isolated influenza virus of the invention. In one embodiment, theinfluenza viruses of the invention may be vaccine vectors for influenzavirus or other pathogens.

A complete virion vaccine may be concentrated by ultrafiltration andthen purified by zonal centrifugation or by chromatography. It isinactivated before or after purification using formalin orbeta-propiolactone, for instance.

A subunit vaccine comprises purified glycoproteins. Such a vaccine maybe prepared as follows: using viral suspensions fragmented by treatmentwith detergent, the surface antigens are purified, byultracentrifugation for example. The subunit vaccines thus containmainly HA protein, and also NA. The detergent used may be cationicdetergent for example, such as hexadecyl trimethyl ammonium bromide(Bachmeyer, 1975), an anionic detergent such as ammonium deoxycholate(Laver & Webster, 1976); or a nonionic detergent such as thatcommercialized under the name TRITON X100. The hemagglutinin may also beisolated after treatment of the virions with a protease such asbromelin, then purified by a method such as that described by Grand andSkehel (1972).

A split vaccine comprises virions which have been subjected to treatmentwith agents that dissolve lipids. A split vaccine can be prepared asfollows: an aqueous suspension of the purified virus obtained as above,inactivated or not, is treated, under stirring, by lipid solvents suchas ethyl ether or chloroform, associated with detergents. Thedissolution of the viral envelope lipids results in fragmentation of theviral particles. The aqueous phase is recuperated containing the splitvaccine, constituted mainly of hemagglutinin and neuraminidase withtheir original lipid environment removed, and the core or itsdegradation products. Then the residual infectious particles areinactivated if this has not already been done.

Inactivated Vaccines.

Inactivated influenza virus vaccines are provided by inactivatingreplicated virus using known methods, such as, but not limited to,formalin or β-propiolactone treatment. Inactivated vaccine types thatcan be used in the invention can include whole-virus (WV) vaccines orsubvirion (SV) (split) vaccines. The WV vaccine contains intact,inactivated virus, while the SV vaccine contains purified virusdisrupted with detergents that solubilize the lipid-containing viralenvelope, followed by chemical inactivation of residual virus.

In addition, vaccines that can be used include those containing theisolated HA and NA surface proteins, which are referred to as surfaceantigen or subunit vaccines.

Live Attenuated Virus Vaccines.

Live, attenuated influenza virus vaccines can be used for preventing ortreating influenza virus infection. Attenuation may be achieved in asingle step by transfer of attenuated genes from an attenuated donorvirus to a replicated isolate or reassorted virus according to knownmethods (see, e.g., Murphy, 1993). Since resistance to influenza A virusis mediated primarily by the development of an immune response to the HAand/or NA glycoproteins, the genes coding for these surface antigensmust come from the reassorted viruses or clinical isolates. Theattenuated genes are derived from the attenuated parent. In thisapproach, genes that confer attenuation preferably do not code for theHA and NA glycoproteins.

Viruses (donor influenza viruses) are available that are capable ofreproducibly attenuating influenza viruses, e.g., a cold adapted (ca)donor virus can be used for attenuated vaccine production. Live,attenuated reassortant virus vaccines can be generated by mating the cadonor virus with a virulent replicated virus. Reassortant progeny arethen selected at 25° C., (restrictive for replication of virulentvirus), in the presence of an appropriate antiserum, which inhibitsreplication of the viruses bearing the surface antigens of theattenuated ca donor virus. Useful reassortants are: (a) infectious, (b)attenuated for seronegative non-adult mammals and immunologically primedadult mammals, (c) immunogenic and (d) genetically stable. Theimmunogenicity of the ca reassortants parallels their level ofreplication. Thus, the acquisition of the six transferable genes of theca donor virus by new wild-type viruses has reproducibly attenuatedthese viruses for use in vaccinating susceptible mammals both adults andnon-adult.

Other attenuating mutations can be introduced into influenza virus genesby site-directed mutagenesis to rescue infectious viruses bearing thesemutant genes. Attenuating mutations can be introduced into non-codingregions of the genome, as well as into coding regions. Such attenuatingmutations can also be introduced into genes other than the HA or NA,e.g., the PB2 polymerase gene (Subbarao et al., 1993). Thus, new donorviruses can also be generated bearing attenuating mutations introducedby site-directed mutagenesis, and such new donor viruses can be used inthe production of live attenuated reassortants vaccine candidates in amanner analogous to that described above for the ca donor virus.Similarly, other known and suitable attenuated donor strains can bereassorted with influenza virus to obtain attenuated vaccines suitablefor use in the vaccination of mammals (Enami et al., 1990; Muster etal., 1991; Subbarao et al., 1993).

It is preferred that such attenuated viruses maintain the genes from thevirus that encode antigenic determinants substantially similar to thoseof the original clinical isolates. This is because the purpose of theattenuated vaccine is to provide substantially the same antigenicity asthe original clinical isolate of the virus, while at the same timelacking pathogenicity to the degree that the vaccine causes minimalchance of inducing a serious disease condition in the vaccinated mammal.

The virus can thus be attenuated or inactivated, formulated andadministered, according to known methods, as a vaccine to induce animmune response in an animal, e.g., a mammal. Methods are well-known inthe art for determining whether such attenuated or inactivated vaccineshave maintained similar antigenicity to that of the clinical isolate orhigh growth strain derived therefrom. Such known methods include the useof antisera or antibodies to eliminate viruses expressing antigenicdeterminants of the donor virus; chemical selection (e.g., amantadine orrimantidine); HA and NA activity and inhibition; and nucleic acidscreening (such as probe hybridization or PCR) to confirm that donorgenes encoding the antigenic determinants (e.g., HA or NA genes) are notpresent in the attenuated viruses. See, e.g., Robertson et al., 1988;Kilbourne, 1969; Aymard-Henry et al., 1985; Robertson et al., 1992.

Pharmaceutical Compositions

Pharmaceutical compositions of the present invention, suitable forinoculation, e.g., nasal, parenteral or oral administration, compriseone or more influenza virus isolates, e.g., one or more attenuated orinactivated influenza viruses, a subunit thereof, isolated protein(s)thereof, and/or isolated nucleic acid encoding one or more proteinsthereof, optionally further comprising sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. The compositions can furthercomprise auxiliary agents or excipients, as known in the art. See, e.g.,Berkow et al., 1987; Avery's Drug Treatment, 1987; Osol, 1980. Thecomposition of the invention is generally presented in the form ofindividual doses (unit doses).

Conventional vaccines generally contain about 0.1 to 200 μg, e.g., 30 to100 μg, of HA from each of the strains entering into their composition.The vaccine forming the main constituent of the vaccine composition ofthe invention may comprise a single influenza virus, or a combination ofinfluenza viruses, for example, at least two or three influenza viruses,including one or more reassortant(s).

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and/or emulsions, which may containauxiliary agents or excipients known in the art. Examples of non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oils suchas olive oil, and injectable organic esters such as ethyl oleate.Carriers or occlusive dressings can be used to increase skinpermeability and enhance antigen absorption. Liquid dosage forms fororal administration may generally comprise a liposome solutioncontaining the liquid dosage form. Suitable forms for suspendingliposomes include emulsions, suspensions, solutions, syrups, and elixirscontaining inert diluents commonly used in the art, such as purifiedwater. Besides the inert diluents, such compositions can also includeadjuvants, wetting agents, emulsifying and suspending agents, orsweetening, flavoring, or perfuming agents. See, e.g., Berkow et al.,1992; Avery's, 1987; and Osol, 1980.

When a composition of the present invention is used for administrationto an individual, it can further comprise salts, buffers, adjuvants, orother substances which are desirable for improving the efficacy of thecomposition. For vaccines, adjuvants, substances which can augment aspecific immune response, can be used. Normally, the adjuvant and thecomposition are mixed prior to presentation to the immune system, orpresented separately, but into the same site of the organism beingimmunized. Examples of materials suitable for use in vaccinecompositions are provided in Osol (1980).

Heterogeneity in a vaccine may be provided by mixing replicatedinfluenza viruses for at least two influenza virus strains, such as 2-20strains or any range or value therein. Influenza A virus strains havinga modern antigenic composition are preferred. Vaccines can be providedfor variations in a single strain of an influenza virus, usingtechniques known in the art.

A pharmaceutical composition according to the present invention mayfurther or additionally comprise at least one chemotherapeutic compound,for example, for gene therapy, immunosuppressants, anti-inflammatoryagents or immune enhancers, and for vaccines, chemotherapeuticsincluding, but not limited to, gamma globulin, amantadine, guanidine,hydroxybenzimidazole, interferon-α, interferon-β, interferon-γ, tumornecrosis factor-alpha, thio semicarbarzones, methisazone, rifampin,ribavirin, a pyrimidine analog, a purine analog, foscarnet,phosphonoacetic acid, acyclovir, dideoxynucleosides, a proteaseinhibitor, or ganciclovir.

The composition can also contain variable but small quantities ofendotoxin-free formaldehyde, and preservatives, which have been foundsafe and not contributing to undesirable effects in the organism towhich the composition is administered.

Pharmaceutical Purposes

The administration of the composition (or the antisera that it elicits)may be for either a “prophylactic” or “therapeutic” purpose. Whenprovided prophylactically, the compositions of the invention which arevaccines are provided before any symptom or clinical sign of a pathogeninfection becomes manifest. The prophylactic administration of thecomposition serves to prevent or attenuate any subsequent infection.When provided prophylactically, the gene therapy compositions of theinvention, are provided before any symptom or clinical sign of a diseasebecomes manifest. The prophylactic administration of the compositionserves to prevent or attenuate one or more symptoms or clinical signsassociated with the disease.

When provided therapeutically, an attenuated or inactivated viralvaccine is provided upon the detection of a symptom or clinical sign ofactual infection. The therapeutic administration of the compound(s)serves to attenuate any actual infection. See, e.g., Berkow et al.,1992; and Avery, 1987. When provided therapeutically, a gene therapycomposition is provided upon the detection of a symptom or clinical signof the disease. The therapeutic administration of the compound(s) servesto attenuate a symptom or clinical sign of that disease.

Thus, an attenuated or inactivated vaccine composition of the presentinvention may be provided either before the onset of infection (so as toprevent or attenuate an anticipated infection) or after the initiationof an actual infection. Similarly, for gene therapy, the composition maybe provided before any symptom or clinical sign of a disorder or diseaseis manifested or after one or more symptoms are detected.

A composition is said to be “pharmacologically acceptable” if itsadministration can be tolerated by a recipient mammal. Such an agent issaid to be administered in a “therapeutically effective amount” if theamount administered is physiologically significant. A composition of thepresent invention is physiologically significant if its presence resultsin a detectable change in the physiology of a recipient patient, e.g.,enhances at least one primary or secondary humoral or cellular immuneresponse against at least one strain of an infectious influenza virus.

The “protection” provided need not be absolute, i.e., the influenzainfection need not be totally prevented or eradicated, if there is astatistically significant improvement compared with a control populationor set of mammals. Protection may be limited to mitigating the severityor rapidity of onset of symptoms or clinical signs of the influenzavirus infection.

Pharmaceutical Administration

A composition of the present invention may confer resistance to one ormore pathogens, e.g., one or more influenza virus strains, by eitherpassive immunization or active immunization. In active immunization, aninactivated or attenuated live vaccine composition is administeredprophylactically to a host (e.g., a mammal), and the host's immuneresponse to the administration protects against infection and/ordisease. For passive immunization, the elicited antisera can berecovered and administered to a recipient suspected of having aninfection caused by at least one influenza virus strain. A gene therapycomposition of the present invention may yield prophylactic ortherapeutic levels of the desired gene product by active immunization.

In one embodiment, the vaccine is provided to a mammalian female (at orprior to pregnancy or parturition), under conditions of time and amountsufficient to cause the production of an immune response which serves toprotect both the female and the fetus or newborn (via passiveincorporation of the antibodies across the placenta or in the mother'smilk).

The present invention thus includes methods for preventing orattenuating a disorder or disease, e.g., an infection by at least onestrain of pathogen. As used herein, a vaccine is said to prevent orattenuate a disease if its administration results either in the total orpartial attenuation (i.e., suppression) of a clinical sign or conditionof the disease, or in the total or partial immunity of the individual tothe disease. As used herein, a gene therapy composition is said toprevent or attenuate a disease if its administration results either inthe total or partial attenuation (i.e., suppression) of a clinical signor condition of the disease, or in the total or partial immunity of theindividual to the disease.

At least one influenza virus isolate of the present invention, includingone which is inactivated or attenuated, one or more isolated viralproteins thereof, one or more isolated nucleic acid molecules encodingone or more viral proteins thereof, or a combination thereof, may beadministered by any means that achieve the intended purposes.

For example, administration of such a composition may be by variousparenteral routes such as subcutaneous, intravenous, intradermal,intramuscular, intraperitoneal, intranasal, oral or transdermal routes.Parenteral administration can be accomplished by bolus injection or bygradual perfusion over time.

A typical regimen for preventing, suppressing, or treating an influenzavirus related pathology, comprises administration of an effective amountof a vaccine composition as described herein, administered as a singletreatment, or repeated as enhancing or booster dosages, over a period upto and including between one week and about 24 months, or any range orvalue therein.

According to the present invention, an “effective amount” of acomposition is one that is sufficient to achieve a desired effect. It isunderstood that the effective dosage may be dependent upon the species,age, sex, health, and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment, and the nature of the effectwanted. The ranges of effective doses provided below are not intended tolimit the invention and represent dose ranges.

The dosage of a live, attenuated or killed virus vaccine for an animalsuch as a mammalian adult organism can be from about 10²-10¹⁵, e.g.,10³-10¹², plaque forming units (PFU)/kg, or any range or value therein.The dose of inactivated vaccine can range from about 0.1 to 1000, e.g.,30 to 100 μg, of HA protein. However, the dosage should be a safe andeffective amount as determined by conventional methods, using existingvaccines as a starting point.

The dosage of immunoreactive HA in each dose of replicated virus vaccinecan be standardized to contain a suitable amount, e.g., 30 to 100 μg orany range or value therein, or the amount recommended by governmentagencies or recognized professional organizations. The quantity of NAcan also be standardized, however, this glycoprotein may be labileduring purification and storage.

Compositions and Dosing for Equine Influenza Vaccines

Equine influenza vaccines generally include representative strains ofH7N7 and H3N8 subtypes either as inactivated whole virus or theirsubunits. They provide protection against influenza by inducing antibodyto the surface glycoproteins, in particular to HA, which is essentialfor viral attachment and entry into cells, and/or potentially importantcell-mediated immune responses to other viral proteins. Vaccination ishelpful in preventing influenza but the protection is short-lived (3-4months using conventional inactivated virus vaccines), so the frequencyof vaccination varies according to how often the horse will likely comein contact with the virus (see Table 1). The usual procedure for theprimary course is vaccination with a single dose followed 3 to 6 weekslater with a second dose. Vaccine manufacturers recommend that boostervaccinations be given at 6- to 12-month intervals thereafter.Alternatively, a horse is administered one 1 to 2 ml dose, e.g., viaintramuscular (IM) injection, a second 1 to 2 ml dose 3 to 4 weeks laterat a different injection site, e.g., via IM injection, and optionally athird 1 to 2 ml dose, e.g., IM or intranasal (IN) administration. Each 1to 2 ml dose of vaccine may contain approximately 1-500 billion virusparticles, and preferably 100 billion particles. Horses in contact witha large number of horses, for example, at a boarding stable, trainingcenters, racetracks, shows, and other such events, are often vaccinatedevery 2-3 months. A three-dose primary series has been shown to induce ahigher and more persistent immunity than the recommended two-dose seriesregardless of the age.

Using conventional vaccines, it is advisable to vaccinate young horses,particularly racehorses and other competition horses, at 4 to 6 monthintervals for several years after their primary course of vaccinations.It has been demonstrated that inclusion of an additional boostervaccination between the second and third vaccination recommended by thevaccine manufacturers is of benefit to young horses. An annual boosterwill usually suffice for older horses such as show jumpers and broodmares that have been vaccinated regularly since they were foals.Vaccination in the face of an ongoing outbreak is sometimes practiced,but is not likely to be effective without an interval of at least 7 to10 days before the freshly vaccinated horses are exposed to infection.Equine influenza outbreaks are not seasonal as in man but are frequentlyassociated with sales or race meets where horses from different regionscongregate and mix. It may therefore be advantageous to time additionalbooster vaccinations to be given prior to such events.

Brood mares should be vaccinated in the later stages of pregnancy, butnot later than 2 weeks prior to foaling, to ensure a good supply ofcolostral antibodies for the foal. Foal vaccinations should begin at 3-6months of age, with a booster at 4-7 months, again at 5-8 months, andrepeated every three months if the foal is at high risk of exposure.

TABLE 1 Foals & Foal & Weanlings Weanlings from from non- VaccinatedVaccinated Performance Pleasure Brood- Mares Mares Yearlings HorseHorses mares Influenza 1st Dose: 1st Dose: Every 3- Every 3-5 Annual Atleast inactivated 9 months 6 months 4 months months with semi-injectable 2nd Dose: 2nd Dose: Boosters annual, 10 months 7 months priorto with 1 3rd Dose: 3rd Dose: likely Booster 11-12 months 8 monthsexposure 4-6 Then at 3 Then at 3 weeks month intervals month prepar-intervals tum Influenza 1st Dose: 1st Dose: Every 4- Every 4-6 EveryAnnual intranasal 12 months; has 12 months; 6 months months 4-6 beforecold- been safely has been months breeding adapted administered tosafely live virus foals less than administered 11 months to foals lessthan 11 months

Influenza vaccines may be combined with tetanus or herpesvirus antigensas well as other pathogens, e.g., equine pathogens. The immune responseelicited by tetanus toxoid is much more durable than that induced byinfluenza antigen. In an intensive influenza vaccination program,vaccines containing influenza only are thus preferred.

Levels of antibody (measured by the SRH assay) required for protectionof horses have been identified through vaccination and challenge studiesand from field data. Because the vaccine-induced antibody response to HAin horses is remarkably short-lived, adjuvants such as aluminumhydroxide or carbomer are normally included to enhance the amplitude andduration of the immune response to whole virus vaccines. Subunit equineinfluenza vaccines containing immune stimulating complexes (ISCOMs) arealso immunogenic.

Historically, antigenic content in inactivated vaccines has beenexpressed in terms of chick cell agglutinating (CCA) units of HA andpotency in terms of HI antibody responses induced in guinea pigs andhorses, neither of which yields reproducible results. The single radialdiffusion (SRD) assay is an improved in vitro potency test that measuresthe concentration of immunologically active HA (expressed in terms ofmicrograms of HA) and can be used for in-process testing before theaddition of adjuvant.

The invention will be further described by the following non-limitingexample.

Example

An approximately 36-hour-old Morgan/Friesian colt was referred to thelarge animal hospital at the University of Wisconsin for an evaluationof altered mentation (mental status), first noticed shortly after birth.Parturition had been unobserved, but the foal had been found separatedfrom the mare by a fence at a few hours of age. The foal was ambulatoryand able to nurse when first discovered but showed progressivedisorientation, apparent blindness, and aimless wandering during thefollowing 36-hour period. A SNAP immunoglobulin G (IgG) assay (IdexxLaboratories, Westbrook, Me.) at 24 hours of age had shown an IgGconcentration>800 mg/dL, and a CBC performed at that time was normal.The foal was treated twice with dimethyl sulfoxide 1 g/kg IV, diluted in5% dextrose before referral.

At presentation, the colt wandered aimlessly, bumped into objects, andappeared blind with sluggish but intact pupillary light responses. Whenpositioned under the mare, the foal nursed successfully. Physicalexamination was unremarkable. A CBC and serum biochemistry were normal,including a serum IgG concentration of 937 mg/dL measured byradioimmunodiffusion.

Initial treatment for presumptive hypoxemic, ischemic encephalopathyincluded a 250 mL loading dose of 20% magnesium sulfate for 1 hour,followed by a constant rate infusion at 42 mL/h and thiaminehydrochloride 2.2 mg/kg IV q24 h. Antimicrobial therapy consisted ofamikacin 20 mg/kg IV q24 h and procaine penicillin G 22,000 U/kg IM q12h. Omeprazole 1 mg/kg PO q24 h also was administered to the foal to helpprevent the development of gastric ulcers.

The foal's mental status remained static during the next 24 hours, andadditional treatment with mannitol 1 g/kg IV q24 h and dexamethasonesodium phosphate 0.1 mg/kg IV q24 h on days 2 and 3 of hospitalizationwas not associated with improvement. On day 3, the foal underwentgeneral anesthesia for a computerized tomographic scan of the skull andproximal spine, which was normal. A cerebrospinal fluid sample wasobtained from the lumbosacral space and was normal on cytologicevaluation and had a normal protein concentration.

On day 4 of hospitalization, the foal developed a right-sided head tiltbut otherwise remained static through day 5 of hospitalization.Magnesium sulfate therapy was discontinued on day 5, but the remainderof the therapeutic regimen was unchanged. On day 6, the foal had 2brief, generalized seizures that were controlled with midazolam 0.05mg/kg IV. Between seizures, the foal was still bright, afebrile, andnursing.

On day 7 of hospitalization, the foal became febrile (40° C.) anddeveloped a mucopurulent nasal discharge and progressive tachypnea withdiffuse adventitious crackles and wheezes on auscultation. Fever,mucopurulent nasal discharge, and coughing had been noted in severalother mares and foals in the neonatal care unit during the previous 7days. Antimicrobial therapy was changed to ticarcillin/clavulanic acid50 mg/kg IV q8 h had gentamicin 6.6 mg/kg IV q24 h, and the foal wastreated with polyionic fluids, although it was still nursing. Duringdays 8-10, the foal's neurologic status continued to improve, with aresolution of the head tilt and a return to normal mentation, but thetachypnea, dyspnea, and adventitious lung sounds worsened. Thoracicradiography at this time showed a severe, diffuse bronchointerstitialpattern. Aminophylline 0.5 mg/kg IV q12 h by slow infusion and nasalinsufflation of oxygen were instituted on days 9 and 10 ofhospitalization. Serial arterial blood gas analysis identified severehypoxemia (PaO₂, 52 mm Hg), hypercapnia (PaCO₂, 68.4 mm Hg), and reducedoxygen saturation (76%) by the end of day 10. Consequently, the foal wasplaced on a mechanical ventilator. Ventilatory support and totalparenteral nutrition were continued for 48 hours, during which timearterial blood gas values normalized on 100% oxygen. Antimicrobialtherapy was continued as before. When challenged on day 13 by theremoval of ventilatory support, the foal developed severe dyspnea andcyanosis and was euthanized at the owner's request. An aerobic cultureof a transtracheal aspirate obtained on day 13 grew Klebsiellapneumoniae and Escherichia coli resistant to ticarcillin/clavulanic acidand gentamicin.

A complete gross and histopathologic postmortem examination wasperformed, as well as a real-time quantitative polymerase chain reaction(PCR) evaluation for the presence of equine herpes virus (EHV)-1 andEHV-4 in samples of nasal secretions; serologic tests to determine ifthere was exposure to equine viral arteritis virus; and a Directigen FluA assay (Bectin Dickinson and Co., Franklin, N.J.) and virus isolationfrom samples of nasal secretions to test for the presence of influenzavirus. Samples of nasal secretions were collected with Dacron swabs thatwere subsequently placed in 2 mL of viral transport media containingphosphate-buffered saline, 0.5% bovine serum albumin, and penicillin G,streptomycin, nystatin, and gentamicin. The nasal swab samples werecollected on day 8 of hospitalization. Follow-up evaluations for theinfluenza virus included immunohistochemistry on snap-frozen andformalin-fixed lung, abdominal viscera, and central nervous systemtissues for the presence of influenza nucleoprotein (NP) expression,virus isolation from frozen lung tissue, and viral sequence analyses.Gross post-mortem examination identified severe diffuse interstitialpneumonia and subdural hemorrhage on the caudal ventral surface of thebrain around the pituitary gland but no evidence of sepsis or pathologyin other organs. Histopathologic examination of the lung identifiednecrotizing bronchitis and brochiolitis, diffuse squamous metaplasia,and multifocal interstitial pneumonia. A mild mononuclear infiltratelined the lower airways and, occasionally, areas of alveolar collapseassociated with congestion and exudate. Evaluation of the brain tissuerevealed a mild dilatation of the ventricular system with diffuse whitematter vacuolation, particularly in the cerebellum. Cresyl violetstaining for the presence of myelin was performed on multiple sectionsand showed diminished but present myelin throughout the brain and spinalcord when compared to tissues from an age-matched control stained inparallel. Additional histopathologic abnormalities in the centralnervous system included an apparent absence of the molecular layerwithin the cerebellum. Serologic tests for equine viral arteritis and areal-time PCR assay for EHV-1 and EHV-4 DNA were negative.

The presence of influenza virus in nasal secretions initially wasconfirmed by a positive Directigen assay. Previous studies havedocumented the sensitivity and specificity of this assay when applied toequine nasal secretion samples (Morely et al., 1995 and Chambers et al.,1994). Samples of the nasal swab transport media also were inoculatedinto the allantoic cavity of embryonated chicken eggs and ontoMadin-Darby canine kidney (MDCK) cells growing in 24-well cell cultureplates. Cytopathologic effects consistent with influenza virus growthwere observed in the inoculated MDCK cells, and an agent that caused thehemagglutination of chicken red blood cells was isolated from theinoculated eggs (Palmar et al., 1975). The presence of influenza virusin the MDCK cell cultures was confirmed by the immunocytochemicalstaining (Landolt et al., 2003) of the inoculated cells with an anti-NPmonoclonal antibody (Mab) 68D2 (kindly provided by Dr. YoshihiroKawaoka, University of Wisconsin-Madison School of Veterinary Medicine)with positive (swine influenza virus inoculated) and negative (mockinoculated) control cells included on the same plate. The identity ofthe virus as an H3-subtype equine influenza virus was confirmed byreverse transcription-PCR amplification of the hemagglutinin (HA) genefrom the isolate, with primers described in Olsen et al. (1997),followed by cycle sequencing of the full-length protein coding region ofthe HA gene and pairwise comparisons to viral sequences available inGenBank (DNASTAR software, version 4.0 for Win32, Bestfit, Madison,Wis.). The virus was shown to be derived from the North American lineageof H3 equine influenza viruses by a phylogenetic analysis that used amaximum parsimony bootstrap analysis (PAUP software, version 4.0b6;David Swofford, Smithsonian Institution, Washington, D.C.) of the HAsequence compared to reference virus strains with a fast-heuristicsearch of 1,000 bootstrap replicates. Similar analyses of portions ofthe nucleotide sequences of the nonstructural protein gene (544nucleotides sequenced) and the NP gene (885 nucleotides sequenced)further confirmed the identity of the virus as a North American-lineageequine influenza virus. This virus is now defined asA/Equine/Wisconsin/1/03. FIG. 1 provides sequences for the coding regionof each gene of that virus.

The presence of influenza virus also was assessed in the lungs and othertissues of the foal. Specifically, immunohistochemistry with Mab 68D2showed scattered, widely dispersed areas of influenza virus NPexpression (predominantly localized around airways) in the frozen aswell as the formalin-fixed lung tissue samples. NP expression was notshown in the other viscera or in the central nervous system. Inaddition, influenza virus was isolated in MDCK cells (and confirmed byimmunocytochemistry and HA gene sequencing) from a sample of the frozenlung tissue.

Acute respiratory distress syndrome (ARDS) in neonatal foals has beendocumented as a consequence of bacterial sepsis (Wilkins, 2003; Hoffmanet al., 1993), perinatal EHV-1 (Frymus et al., 1986; Gilkerson et al.,1999) and EHV-4 (Gilkerson et al., 1999), and equine viral arteritisinfection (Del Piero et al., 1997). Less severe lower airway diseaseoccasionally is documented with adenovirus and EHV-2 infections,particularly in the immunocompromised patient (Webb et al., 1981; Murrayet al., 1996). Bronchointerstitial pneumonia and ARDS are high-mortalityrespiratory diseases of older foals with several potential causes,including bacterial and viral infections (Lakritz et al., 1993). Whetherit occurs in neonates experiencing septic shock or in older foals withdiffuse bronchointerstitial pneumonia, ARDS is characterized byacute-onset, rapidly progressive, severe tachypnea. The increasedrespiratory effort, worsening cyanosis, hypoxemia, and hypercapnia thataccompany ARDS frequently are poorly responsive to aggressive therapy(Wilkins, 2003; Lakritz et al., 1993). It is a category of respiratorydisease with several potential etiologies and a mortality rate thatfrequently exceeds 30% despite intensive treatment with antimicrobials,oxygen, anti-inflammatory agents, brochodilators, and thermoregulatorycontrol. Equine influenza is a well-documented cause of upperrespiratory disease in horses worldwide (Wilkins, 2003; Van Maanen etal., 2002; Wilson, 1993), but very little information exists in theliterature about the manifestations of this disease in neonates. Asingle report describes bronchointerstitial pneumonia in a 7-day-oldfoal from which equine influenza A was isolated (Britton et al., 2002);this foal resembles the foal described herein.

The foal detailed in this study was one of several hospitalized horsesthat developed fever, mucopurulent nasal discharge, and coughing duringa 2- or 3-week period. Clinical signs in the other affected horses,including high-risk neonates, generally were confined to the upperrespiratory tract, except for mild systemic signs of fever andinappetance. The reason for the severity of the pulmonary failure inthis foal is unclear. Treatment did include the potentiallyimmunosuppressive drug dexamethasone and general anesthesia for adiagnostic procedure, both of which may have predisposed the foal to thedevelopment of pneumonia. The impact of the foal's neurologic disease onthe development and progression of respiratory disease also is unclear.The histologic findings of diffuse vacuolization, decreased myelinthroughout the central nervous system, and absent molecular layer withinthe cerebellum do not fit any specific clinical or histopathologicdiagnosis. The foal could have had impaired central control ofrespiration, because the areas of the brain involved in the control ofrespiration (the pons and medulla oblongata) showed diffusevacuolization and diminished myelin staining. Any subsequent impairmentof ventilation would likely have been a terminal event given thenormalcy of ventilatory function until several days afterhospitalization. However, the abnormal mentation from birth, thevacuolization, the decreased myelinization in the central nervoussystem, and the cerebellar abnormalities are suggestive of a concurrent,congenital neurologic abnormality, which may have compromised the foal'sability to respond to worsening respiratory function. The focalhemorrhage observed on the caudal ventral aspect of the brain was mildand was possibly a consequence of trauma during one of the seizures thefoal experienced.

The mare had been vaccinated semiannually against influenza for the past2 years with a killed product and was given a booster vaccination inlate pregnancy. Considering the evidence of adequate passive transfer inthis foal, these antibodies apparently did not confer adequateprotection for the foal. Furthermore, phylogenetic analysis of theisolate obtained from the foal characterized it as an H3N8 subtype, andthe commercial product used to vaccinate the mare in late pregnancycontained an influenza virus strain of the same subtype, suggesting thatpassive transfer cannot be guaranteed to protect against naturalinfection under certain circumstances. This lack of vaccine efficacy isconsistent with a recent study by Mumford et al. (2003) that describesthe failure of commercially available H7N7 and H3N8 equine influenzavirus vaccines to protect adults against clinical respiratory diseasethat results from a natural infection with certain H3N8 virus strains.The transtracheal recovery of 2 bacterial species that were resistant tothe antimicrobial regimen in place at the time of death confounds theconclusion that influenza was the sole cause of death. However,postmortem examination identified no gross or histopathologic evidenceof sepsis, and synergism occurs between the influenza virus and somebacterial pathogens, combining to cause pneumonia with increasedmortality (McCullers et al., 2003; Simonsen, 1999). Furthermore, theisolation of the infectious virus and the immunohistochemicaldemonstration of viral antigen from the lung tissue obtained postmortem,6 days after the virus initially was recovered by a nasopharyngeal swab,provide strong evidence of a pathologic contribution from influenzavirus in this foal's respiratory failure.

To compare the growth characteristics of avian, equine, human, andporcine lineage viruses in primary canine respiratory epithelial cellsand to investigate the species influence on their growthcharacteristics, cultured cells were infected at an MOI of 3 withviruses including A/Equine/Wisconsin/1/03 and incubated for up to 10hours. The other viruses included six human and swine influenza A virusisolates (A/Phillipines/08/98, A/Panama/2002/99, A/Costa Rica/07/99;A/Swine/NorthCarolina/44173/00, A/Swine/Minnesota/593/99,A/Swine/Ontario/00130/97, and two equine influenza viruses(A/Equine/Kentucky/81 and A/Equine/Kentucky/91). At the end of theexperiment, the cells were formalin fixed for immunocytochemistry andflow cytometry analyses.

The six human and swine influenza virus isolates mentioned above readilyinfected substantially all (80-90%) of the canine respiratory epithelialcells and grew to high titers (10⁵³-10⁷ TCID₅₀/ml) in those cells.A/Equine/Kentucky/81 and A/Equine/Kentucky/91 were highly restricted intheir infectivity (<10% of the cells infected) with little (10^(1.7)TCID₅₀/ml for A/Equine/Kentucky/81) or no (for A/Equine/Kentucky/91)detectable viral growth. In contrast, A/Equine/Wisconsin/1/03 infected alarger percentage (about 30%) of the primary canine respiratoryepithelial cells and grew to substantially higher titers (about 10^(4.8)TCID₅₀/ml) in those cells. The results demonstrated that all influenza Aviruses tested were able to infect canine primary respiratory epithelialcells. However, the infectivity and replication characteristics of theviruses were strongly lineage-dependent.

Dubovi et al. (2004) noted recurrent outbreaks of severe respiratorydisease characterized by coughing and fever in greyhound dogs at racingkennels in Florida. Most affected dogs recovered, but some succumbed toa fatal hemorrhagic pneumonia. Lung tissues from 5 of the dogs that diedfrom the hemorrhagic pneumonia syndrome were subjected to virusisolation studies in African green monkey kidney epithelial cells(Vero), Madin-Darby canine kidney epithelial cells (MDCK), primarycanine kidney epithelial cells, primary canine lung epithelial cells,primary bovine testicular epithelial cells, canine tumor fibroblasts(A-72), and human colorectal adenocarcinoma epithelial cells (HRT-18)(Dubovi et al., 2004). Cytopathology in the MDCK cells was noted on thefirst passage of lung homogenate from one of the dogs, and the loss ofcytopathology upon subsequent passage to cells cultured without trypsincoupled with the presence of hemagglutinating activity in culturesupernatants suggested the presence of an influenza virus (Dubovi etal., 2004). The virus was initially identified as influenza virus by PCRusing primers specific for the matrix gene. The canine influenza virushas been designated as the A/Canine/Florida/43/04 strain. Based on virusisolation from the lungs, the presence of viral antigens in lung tissuesby immunohistochemistry, and seroconversion data, Dubovi et al. (2004)concluded that the isolated influenza virus was most likely theetiological agent responsible for the fatal hemorrhagic pneumonia inracing greyhounds during the Jacksonville 2004 outbreak, and that thiswas the first report of an equine influenza virus associated withrespiratory disease in dogs (Dubovi et al., 2004). The HA protein of thecanine isolate differs from the A/Equine/Wisconsin/1/03 strain by only 6amino acids.

REFERENCES

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All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

1. (canceled)
 2. A method of preparing a vaccine, comprising providingan isolated live H3 influenza virus comprising a gene segment withsequences for a HA having SEQ ID NO: 1 or a HA having at least 98% aminoacid sequence identity to SEQ ID NO: 1 which HA does not have a valineat position 78 or an asparagine at position 159; combining the isolatedH3 influenza virus with an adjuvant or treating the isolated H3influenza virus with an agent that inactivates the virus, therebyproviding a composition; and providing an individual dose of thecomposition comprising an amount effective to immunize a mammal.
 3. Themethod of claim 2 wherein the mammal is a horse.
 4. The method of claim2 wherein the mammal is a canine.
 5. The method of claim 2 wherein thecomposition comprises inactivated virus.
 6. The method of claim 2wherein the H3 influenza virus comprises at least one of the followinggene segments: a gene segment with sequences for a NA having SEQ ID NO:2or having at least 95% amino acid sequence identity to SEQ ID NO:2, agene segment with sequences for a PB1 having SEQ ID NO:3 or having atleast 95% amino acid sequence identity to SEQ ID NO:3, a gene segmentwith sequences for a PB2 having SEQ ID NO:4 or having at least 95% aminoacid sequence identity to SEQ ID NO:4, a gene segment with sequences fora PA having SEQ ID NO:5 or having at least 95% amino acid sequenceidentity to SEQ ID NO:5, a gene segment with sequences for a NP havingSEQ ID NO:6 or having at least 95% amino acid sequence identity to SEQID NO:6, a gene segment with sequences for a M1 having SEQ ID NO:7 orhaving at least 95% amino acid sequence identity to SEQ ID NO:7, a genesegment with sequences for a M2 having SEQ ID NO: 17 or having at least95% amino acid sequence identity to SEQ ID NO: 17, a gene segment withsequences for a NS1 having SEQ ID NO:8 or having at least 95% amino acidsequence identity to SEQ ID NO:8, or a gene segment with sequences for aNS2 having SEQ ID NO: 18 or having at least 95% amino acid sequenceidentity to SEQ ID NO:18.
 7. The method of claim 2 wherein thecomposition further comprises a different isolated influenza virus. 8.The method of claim 2 wherein the composition further comprises apathogen other than the H3 influenza virus.
 9. The method of claim 2wherein the composition comprises the adjuvant.
 10. The method of claim2 further comprises a pharmaceutically acceptable carrier.
 11. Themethod of claim 10 wherein the carrier is suitable for intramuscularadministration.
 12. The method of claim 2 wherein the composition isfreeze-dried.
 13. A method of preparing a vaccine, comprising providingan isolated live H3 influenza virus comprising a gene segment withsequences for a HA having SEQ ID NO:1 or a HA having at least 95% aminoacid sequence identity to SEQ ID NO: 1 which HA has an alanine atposition 78 and a serine at position 159; combining the isolated H3influenza virus with an adjuvant or treating the isolated H3 influenzavirus with an agent that inactivates the virus, thereby providing acomposition; and providing an individual dose of the compositioncomprising an amount effective to immunize a mammal.
 14. The method ofclaim 13 wherein the mammal is a horse.
 15. The method of claim 13wherein the mammal is a canine.
 16. The method of claim 13 wherein thecomposition comprises inactivated virus.
 17. The method of claim 13wherein the carrier is suitable for intranasal administration.
 18. Themethod of claim 13 wherein the composition further comprises a differentisolated influenza virus.
 19. The method of claim 13 wherein thecomposition further comprises a pathogen other than the H3 influenzavirus.
 20. The method of claim 13 wherein the composition comprises theadjuvant.
 21. The method of claim 13 further comprises apharmaceutically acceptable carrier suitable for intramuscular orintransasl administration.